A major effect of methotrexate and related drugs is inhibition of DNA synthesis, by depression (indirectly or directly) of the activity of the enzyme, thymidylate synthetase, resulting in diminished levels of the DNA precursor, dTTP, and enhanced deoxyuridylate. The proposed research will attempt to determine whether (or to what extent) deoxyuridylate incorporation into DNA, and subsequent removal, takes place in intact cells as a result of the alterations in precursor pools caused by methotrexate. The studies will be carried out primarily on cultured human lymphoblasts and will seek evidence for deoxyuridylate incorporation/removal that will include: a. Uracil in DNA; b. apyrimidinic sites in DNA; c. deoxyuridylate removal products, i.e., uracil, and the excision products from the apyrimidinic sites; d. size of newly synthesized DNA; e. cellular levels of dUTP. It is hoped that one of the results of these studies will be to provide evidence concerning the mechanism of cell death by methotrexate and related drugs, and information to improve the chemotherapeutic effectiveness of these agents.